Mutagenesis, 2016, 31, 69–81 doi:10.1093/mutage/gev055 Original Manuscript Advance Access publication 4 August 2015
Original Manuscript
Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform Kana Nishihara1,2, Ruili Huang2, Jinghua Zhao2, Sampada A. Shahane2, Kristine L. Witt3, Stephanie L. Smith-Roe3, Raymond R. Tice3, Shunichi Takeda1 and Menghang Xia2,* Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Kyoto 606-8501, Japan, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and 3Division of the National Toxicology Program, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, National Institutes of Health, Research Triangle Park, NC 27709, USA 1 2
*To whom correspondence should be addressed. Tel: +1 301 217 5718; Fax: +1 301 217 5736; Email: [email protected] Received 5 February 2015; Revised 11 June 2015; Accepted 10 July 2015.
Abstract DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70−/−/RAD54−/− and REV3−/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many wellknown genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.
Introduction Genotoxic chemicals can generate a variety of DNA lesions, such as single-strand DNA breaks, double-strand DNA breaks (DSBs),
alkylation of DNA bases and covalent links between bases [intrastrand and interstrand crosslinks (ICLs)]. Damage left unrepaired or repaired incorrectly might lead to genetic mutations and/or instability and increase the risk of carcinogenesis (1).
Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2015.
69
K. Nishihara et al., 2016, Vol. 31, No. 1
70 To reduce the risk of exposure to toxic chemicals, newly developed chemicals and established chemicals that have not been studied previously require comprehensive toxicological characterisation, including an assessment of genotoxic potential. Traditionally, in vitro, chemical-induced DNA damage has been assessed by a battery of independent assays, including the Ames test, mouse lymphoma assay, micronucleus test, chromosomal aberration test and/or the comet assay (2). However, none of these assays is well-suited for high-throughput screening (HTS) using large chemical libraries on a robotic platform because of their complexity and specific protocol features, including duration of exposure, number of cells per sample needed and the use of liver S9 mix for metabolic activation. To overcome the limitations of traditional genotoxicity tests for adaption to HTS platforms and to increase testing throughput, we previously developed and optimised a new screening system using a group of DNA repair-deficient chicken B-lymphocyte DT40 cell lines on a quantitative HTS (qHTS) platform (3), as part of the US Tox21 program (4,5). With this screening system, we measured differential cytotoxicity using a cell viability assay (CellTiter-Glo) that measures intracellular adenosine triphosphate (ATP) levels in wild-type versus six different DNA repair-deficient DT40 cell lines (ATM−/−, FANCC−/−, POLβ−/−, KU70−/−/RAD54−/−, REV3−/− and UBC13−/−), following exposure to a library of 1408 compounds. We observed that the combination of the KU70−/−/RAD54−/− and REV3−/− cell lines provided the highest sensitivity to known genotoxic chemicals, such as actinomycin D, adriamycin, alachlor, benzotrichloride and melphalan, compared with any other combination of DNA repair-deficient clones (3). In the present study, we screened the Tox21 10K compound library against the KU70−/−/RAD54−/− and REV3−/− DT40 cell lines and the parental wild-type cell line using the same cell viability assay described previously (3). In this assay system, active (i.e. genotoxic) compounds are those that reduce cell proliferation to a greater extent in the DNA repair-deficient cell lines compared with the parental, isogenic wild-type cell line (6). KU70 and RAD54 participate in DSB repair by non-homologous end joining (NHEJ) and homologous recombination (HR), respectively (7,8). REV3 is the catalytic subunit of translesion DNA synthesis (TLS) polymerase ζ (9,10), can bypass a wide variety of DNA lesions to maintain progression of DNA replication (11), and may play a dominant role in TLS-mediated mutagenesis in mammalian cells (12). In addition to TLS, REV3 may operate within the Fanconi anemia DNA-repair pathway to eliminate ICLs (13,14).
In the primary screening of the Tox21 10K compound library, we identified several well-known genotoxic compounds (e.g. adriamycin, melphalan) that induced significantly greater cytotoxicity in the DNA repair-deficient cell lines compared with wild-type cell line. Moreover, several compounds previously untested for genotoxicity were identified as potential direct-acting genotoxicants in our assay. In follow-up studies, selected compounds were evaluated further for genotoxicity using a high content micronucleus (MN) assay and phosphorylated H2AX (γH2AX) immunostaining. Using this approach (Figure 1), we confirmed several known and novel genotoxic chemicals. The results presented in this study demonstrate the utility of this approach for evaluating the genotoxic activity of chemicals in a qHTS format and for acquiring information on the type(s) of DNA damage induced by these chemicals.
Materials and methods Tox21 10K compound library and chemicals The Tox21 10K compound library containing >8300 unique compounds has been previously described (4). For the follow-up studies, adriamycin [Chemical Abstract Services Registry Number (CASRN) = 25316-40-9], cyclophosphamide (CASRN = 6055-19-2), melphalan (CASRN = 148-82-3), mitomycin C (CASRN = 50-07-7), sobuzoxane (CASRN = 9863195-9), tetraoctylammonium bromide (CASRN = 14866-33-2), tetraphenylolethane glycidyl ether (CASRN = 7328-97-4), trifluridine (CASRN = 70-00-8) and 2-oxiranemethanamine (CASRN = 2876832-3) were purchased from Sigma–Aldrich (St Louis, MO, USA). AD-67 (CASRN = 71526-07-3) was obtained from Ark Pharm (Libertyville, IL, USA). 4-Hydroperoxy cyclophosphamide (CASRN = 39800-16-3) was obtained from Toronto Research Chemicals (North York, ON, Canada). All chemicals were dissolved in dimethyl sulfoxide (DMSO, Fischer Scientific, Pittsburgh, PA, USA) and prepared as 20 mM stock solutions prior to use.
Cell culture DNA repair-deficient DT40 cell lines, developed at Kyoto University, Japan (8,11,15), and the isogenic wild-type cell line were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA), 1% chicken serum (Life Technologies), 50 µM β-mercaptoethanol (Sigma–Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies).
Figure 1. Flow chart for the identification of genotoxic compounds. One hundred and nineteen compounds with ≥3-fold increase in cytotoxicity (P
Original Manuscript
Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform Kana Nishihara1,2, Ruili Huang2, Jinghua Zhao2, Sampada A. Shahane2, Kristine L. Witt3, Stephanie L. Smith-Roe3, Raymond R. Tice3, Shunichi Takeda1 and Menghang Xia2,* Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Kyoto 606-8501, Japan, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and 3Division of the National Toxicology Program, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, National Institutes of Health, Research Triangle Park, NC 27709, USA 1 2
*To whom correspondence should be addressed. Tel: +1 301 217 5718; Fax: +1 301 217 5736; Email: [email protected] Received 5 February 2015; Revised 11 June 2015; Accepted 10 July 2015.
Abstract DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70−/−/RAD54−/− and REV3−/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many wellknown genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.
Introduction Genotoxic chemicals can generate a variety of DNA lesions, such as single-strand DNA breaks, double-strand DNA breaks (DSBs),
alkylation of DNA bases and covalent links between bases [intrastrand and interstrand crosslinks (ICLs)]. Damage left unrepaired or repaired incorrectly might lead to genetic mutations and/or instability and increase the risk of carcinogenesis (1).
Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2015.
69
K. Nishihara et al., 2016, Vol. 31, No. 1
70 To reduce the risk of exposure to toxic chemicals, newly developed chemicals and established chemicals that have not been studied previously require comprehensive toxicological characterisation, including an assessment of genotoxic potential. Traditionally, in vitro, chemical-induced DNA damage has been assessed by a battery of independent assays, including the Ames test, mouse lymphoma assay, micronucleus test, chromosomal aberration test and/or the comet assay (2). However, none of these assays is well-suited for high-throughput screening (HTS) using large chemical libraries on a robotic platform because of their complexity and specific protocol features, including duration of exposure, number of cells per sample needed and the use of liver S9 mix for metabolic activation. To overcome the limitations of traditional genotoxicity tests for adaption to HTS platforms and to increase testing throughput, we previously developed and optimised a new screening system using a group of DNA repair-deficient chicken B-lymphocyte DT40 cell lines on a quantitative HTS (qHTS) platform (3), as part of the US Tox21 program (4,5). With this screening system, we measured differential cytotoxicity using a cell viability assay (CellTiter-Glo) that measures intracellular adenosine triphosphate (ATP) levels in wild-type versus six different DNA repair-deficient DT40 cell lines (ATM−/−, FANCC−/−, POLβ−/−, KU70−/−/RAD54−/−, REV3−/− and UBC13−/−), following exposure to a library of 1408 compounds. We observed that the combination of the KU70−/−/RAD54−/− and REV3−/− cell lines provided the highest sensitivity to known genotoxic chemicals, such as actinomycin D, adriamycin, alachlor, benzotrichloride and melphalan, compared with any other combination of DNA repair-deficient clones (3). In the present study, we screened the Tox21 10K compound library against the KU70−/−/RAD54−/− and REV3−/− DT40 cell lines and the parental wild-type cell line using the same cell viability assay described previously (3). In this assay system, active (i.e. genotoxic) compounds are those that reduce cell proliferation to a greater extent in the DNA repair-deficient cell lines compared with the parental, isogenic wild-type cell line (6). KU70 and RAD54 participate in DSB repair by non-homologous end joining (NHEJ) and homologous recombination (HR), respectively (7,8). REV3 is the catalytic subunit of translesion DNA synthesis (TLS) polymerase ζ (9,10), can bypass a wide variety of DNA lesions to maintain progression of DNA replication (11), and may play a dominant role in TLS-mediated mutagenesis in mammalian cells (12). In addition to TLS, REV3 may operate within the Fanconi anemia DNA-repair pathway to eliminate ICLs (13,14).
In the primary screening of the Tox21 10K compound library, we identified several well-known genotoxic compounds (e.g. adriamycin, melphalan) that induced significantly greater cytotoxicity in the DNA repair-deficient cell lines compared with wild-type cell line. Moreover, several compounds previously untested for genotoxicity were identified as potential direct-acting genotoxicants in our assay. In follow-up studies, selected compounds were evaluated further for genotoxicity using a high content micronucleus (MN) assay and phosphorylated H2AX (γH2AX) immunostaining. Using this approach (Figure 1), we confirmed several known and novel genotoxic chemicals. The results presented in this study demonstrate the utility of this approach for evaluating the genotoxic activity of chemicals in a qHTS format and for acquiring information on the type(s) of DNA damage induced by these chemicals.
Materials and methods Tox21 10K compound library and chemicals The Tox21 10K compound library containing >8300 unique compounds has been previously described (4). For the follow-up studies, adriamycin [Chemical Abstract Services Registry Number (CASRN) = 25316-40-9], cyclophosphamide (CASRN = 6055-19-2), melphalan (CASRN = 148-82-3), mitomycin C (CASRN = 50-07-7), sobuzoxane (CASRN = 9863195-9), tetraoctylammonium bromide (CASRN = 14866-33-2), tetraphenylolethane glycidyl ether (CASRN = 7328-97-4), trifluridine (CASRN = 70-00-8) and 2-oxiranemethanamine (CASRN = 2876832-3) were purchased from Sigma–Aldrich (St Louis, MO, USA). AD-67 (CASRN = 71526-07-3) was obtained from Ark Pharm (Libertyville, IL, USA). 4-Hydroperoxy cyclophosphamide (CASRN = 39800-16-3) was obtained from Toronto Research Chemicals (North York, ON, Canada). All chemicals were dissolved in dimethyl sulfoxide (DMSO, Fischer Scientific, Pittsburgh, PA, USA) and prepared as 20 mM stock solutions prior to use.
Cell culture DNA repair-deficient DT40 cell lines, developed at Kyoto University, Japan (8,11,15), and the isogenic wild-type cell line were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA), 1% chicken serum (Life Technologies), 50 µM β-mercaptoethanol (Sigma–Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies).
Figure 1. Flow chart for the identification of genotoxic compounds. One hundred and nineteen compounds with ≥3-fold increase in cytotoxicity (P
